1. Field of the Invention
The present invention concerns infectious porcine circovirus type-1 (PCV 1) and type-2 (PCV2) DNA clones, chimeric PCV 1-2 infectious DNA clones and live chimeric viruses derived from the chimeric DNA clones, useful as vaccines.
2. Description of the Related Art
All patents and publications cited in this specification are hereby incorporated by reference in their entirety.
Porcine circovirus (PCV) was originally isolated as a cell culture contaminant of a porcine kidney cell line PK-15 (I. Tischer et al., “A very small porcine virus with circular single-stranded DNA,” Nature 295:64-66 (1982); I. Tischer et al., “Characterization of papovavirus and picornavirus-like particles in permanent pig kidney cell lines,” Zentralbl. Bakteriol. Hyg. Otg. A. 226(2):153-167 (1974)). PCV is a small icosahedral non-enveloped virus that contains a single stranded circular DNA genome of about 1.76 kb. PCV is classified in the family of Circoviridae, which consists of three other animal circoviruses (chicken anemia virus (CAV), psittacine beak and feather disease virus (PBFDV) and the recently discovered columbid circovirus (CoCV) from pigeons) and three plant circoviruses (banana bunchy top virus, coconut foliar decay virus and subterranean clover stunt virus) (M. R. Bassami et al., “Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anemia virus,” Virology 249:453-459 (1998); J. Mankertz et al., “Transcription analysis of porcine circovirus (PCV),” Virus Genes 16:267-276 (1998); A. Mankertz et al., “Cloning and sequencing of columbid circovirus (CoCV), a new circovirus from pigeons,” Arch. Virol. 145:2469-2479 (2000); B. M. Meehan et al., “Sequence of porcine circovirus DNA: affinities with plant circoviruses,” J. Gen. Virol. 78:221-227 (1997); B. M. Meehan et al., “Characterization of novel circovirus DNAs associated with wasting syndromes in pigs,” J. Gen. Virol. 79:2171-2179 (1998); D. Todd et al., “Comparison of three animal viruses with circular single-stranded DNA genomes,” Arch. Virol. 117:129-135 (1991)). Members of the three previously recognized animal circoviruses (PCV, CAV, and PBFDV) do not share nucleotide sequence homology or antigenic determinants with each other (M. R. Bassami et al., 1998, supra; D. Todd et al., 1991, supra). The genome of the newly identified CoCV shared about 40% nucleotide sequence identity with that of PCV (A. Mankertz et al., “Cloning and sequencing of columbid circovirus (CoCV), a new circovirus from pigeons,” Arch. Virol. 145:2469-2479 (2000)). Recently, a novel human circovirus with a circular genome, designated as transfusion transmitted virus or TT virus (TTV), was identified from individuals associated with post-transfusion hepatitis (H. Miyata et al., “Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus,” J. Virol. 73:3582-3586 (1999); T. Nishizawa et al., “A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology,” Biochem. Biophys. Res. Commun. 241:92-97 (1997)). Additionally, a human TTV-like mini virus (TLMV) was identified from normal blood donors (P. Biagini et al., “Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates,” J. Gen. Virol. 82: 379-383 (2001); K. Takahashi et al., “Identification of a new human DNA virus (TTV-like mini virus, TLMV) intermediately related to TT virus and chicken anemia virus,” Arch. Virol. 145:979-93 (2000)) and a third novel human circovirus, known as SEN virus (SENV), was also discovered from humans with post-transfusion hepatitis (T. Umemura et al., “SEN virus infection and its relationship to transfusion-associated hepatitis,” Hepathology 33:1303-1311 (2001)). The genomic organization of both human TTV and TLMV is similar to that of the CAV (P. Biagini et al., 2001, supra; H. Miyata et al., 1999, supra; K. Takahashi et al., 2000, supra). Although antibodies to PCV were found in various animal species including humans, mice, cattle and pigs (G. M. Allan et al., “Production, preliminary characterization and applications of monoclonal antibodies to porcine circovirus,” Vet. Immunol. Immunopathol. 43:357-371 (1994); G. C. Dulac and A. Afshar, “Porcine circovirus antigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies to circovirus in Canadian pigs,” Can. J. Vet. Res. 53:431-433 (1989); S. Edwards and J. J. Sands, “Evidence of circovirus infection in British pigs,” Vet. Rec. 134:680-1 (1994); J. C. Harding and E. G. Clark, “Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS),” Swine Health and Production 5:201-203 (1997); R. K. Hines and P. D. Lukert, “Porcine circovirus: a serological survey of swine in the United States,” Swine Health and Production 3:71-73 (1995); G. P. Nayar et al., “Evidence for circovirus in cattle with respiratory disease and from aborted bovine fetuses,” Can. Vet. J. 40:277-278 (1999); I. Tischer et al., “Distribution of antibodies to porcine circovirus in swine populations of different breeding farms,” Arch. Virol. 140:737-743 (1995); I. Tischer et al., “Presence of antibodies reacting with porcine circovirus in sera of humans, mice, and cattle,” Arch. Virol. 140:1427-1439 (1995)), little is known regarding the pathogenesis of PCV in these animal species. Experimental infection of pigs with the PK-15 cells-derived PCV did not produce clinical disease and thus, this virus is not considered to be pathogenic to pigs (G. M. Allan et al., “Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material,” Vet. Microbiol. 44:49-64 (1995); I. Tischer et al., “Studies on epidemiology and pathogenicity of porcine circovirus,” Arch. Virol. 91:271-276 (1986)). The nonpathogenic PCV derived from the contaminated PK-15 cell line was designated as porcine circovirus type 1 or PCV1.
Postweaning multisystemic wasting syndrome (PMWS), first described in 1991 (J. C. Harding and E. G. Clark, 1997, supra), is a complex disease of weaning piglets that is becoming increasingly more widespread. With the threat of a potential serious economic impact upon the swine industry, it has become urgent to develop a vaccine against PCV2, the primary causative agent of PMWS. PMWS mainly affects pigs between 5-18 weeks of age. Clinical PMWS signs include progressive weight loss, dyspnea, tachypnea, anemia, diarrhea, and jaundice. Mortality rate may vary from 1% to 2%, and up to 40% in some complicated cases in the U.K. (M. Muirhead, “Sources of information on PMWS/PDNS,” Vet. Rec. 150:456 (2002)). Microscopic lesions characteristic of PMWS include granulomatous interstitial pneumonia, lymphadenopathy, hepatitis, and nephritis (G. M. Allan and J. A. Ellis, “Porcine circoviruses: a review,” J. Vet. Diagn. Invest. 12:3-14 (2000); J. C. Harding and E. G. Clark, 1997, supra). PMWS has now been recognized in pigs in Canada, the United States (G. M. Allan et al., “Novel porcine circoviruses from pigs with wasting disease syndromes,” Vet. Rec. 142:467-468 (1998); G. M. Allan et al., “Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe,” J. Vet. Diagn. Invest. 10:3-10 (1998); G. M. Allan and J. A. Ellis, 2000, supra; J. Ellis et al., “Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome,” Can. Vet. J. 39:44-51 (1998); A. L. Hamel et al., “Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs,” J. Virol. 72:5262-5267 (1998); M. Kiupel et al., “Circovirus-like viral associated disease in weaned pigs in Indiana,” Vet. Pathol. 35:303-307 (1998); R. Larochelle et al., “Identification and incidence of porcine circovirus in routine field cases in Quebec as determined by PCR,” Vet. Rec. 145:140-142 (1999); B. M. Meehan et al., 1998, supra; I. Morozov et al., “Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome,” J. Clin. Microbiol. 36:2535-2541 (1998)), most European countries (G. M. Allan et al., “Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe,” J. Vet. Diagn. Invest. 10:3-10 (1998); G. M. Allan and J. A. Ellis, 2000, supra; S. Edwards and J. J. Sands, 1994, supra; S. Kennedy et al., “Porcine circovirus infection in Northern Ireland,” Vet. Rec. 142:495-496 (1998); A. Mankertz et al., “Characterization of PCV-2 isolates from Spain, Germany and France,” Virus Res. 66:65-77 (2000); C. Rosell et al., “Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome. Vet. Rec. 146:40-43 (2000); P. Spillane et al., “Porcine circovirus infection in the Republic of Ireland,” Vet. Rec. 143:511-512 (1998); G. J. Wellenberg et al., “Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in the Netherlands,” Vet. Quart. 22:167-72 (2000)) and some countries in Asia (C. Choi et al., “Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction,” J. Vet. Diagn. Invest. 12:151-153 (2000); A. Onuki et al., “Detection of porcine circovirus from lesions of a pig with wasting disease in Japan,” J. Vet. Med. Sci. 61:1119-1123 (1999)). PMWS potentially has a serious economic impact on the swine industry worldwide.
The primary causative agent of PMWS is a pathogenic strain of PCV designated as porcine circovirus type 2 or PCV2 (G. M. Allan et al., “Novel porcine circoviruses from pigs with wasting disease syndromes,” Vet. Rec. 142:467-468 (1998); G. M. Allan et al., “Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe,” J. Vet. Diagn. Invest. 10:3-10 (1998); G. M. Allan et al., “Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland,” Vet. Microbiol. 66:115-23 (1999); G. M. Allan and J. A. Ellis, 2000, supra; J. Ellis et al., 1998, supra; A. L. Hamel et al., 1998, supra; B. M. Meehan et al., 1998, supra; I. Morozov et al., 1998, supra). The complete genomic sequence of the PMWS-associated PCV2 has been determined (M. Fenaux et al., “Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2,” J. Clin. Microbiol. 38:2494-503 (2000); A. L. Hamel et al., 1998, supra; J. Mankertz et al., 1998, supra; B. M. Meehan et al., 1997, supra; B. M. Meehan et al., 1998, supra; I. Morozov et al., 1998, supra).
PCV1 is ubiquitous in pigs but is not pathogenic to pigs. In contrast, the genetically related PCV2 is pathogenic and causes PMWS in pigs. Sequence analyses reveals that the PMWS-associated PCV2 shares only about 75% nucleotide sequence identity with the nonpathogenic PCV1. The ORF2 gene of both the nonpathogenic PCV1 and the pathogenic PCV2 encodes for the major immunogenic viral capsid protein (P. Nawagitgul et al., “Modified indirect porcine circovirus (PCV) type 2-based and recombinant capsid protein (ORF2)-based ELISA for the detection of antibodies to PCV,” Immunol. Clin. Diagn. Lab Immunol. 1:33-40 (2002); P. Nawagitgul et al., “Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein,” J. Gen. Virol. 81:2281-2287 (2000)).
Initial attempts to reproduce clinical PMWS in conventional pigs by PCV2 inoculation were unsuccessful (M. Balasch et al., “Experimental inoculation of conventional pigs with tissue homogenates from pigs with post-weaning multisystemic wasting syndrome,” J. Comp. Pathol. 121:139-148 (1999); M. Fenaux et al., “Cloned Genomic DNA of Type 2 Porcine Circovirus (PCV-2) Is Infectious When Injected Directly into the Liver and Lymph Nodes of SPF Pigs: Characterization of Clinical Disease, Virus Distribution, and Pathologic Lesions,” J. Virol. 76:541-551 (2002)). Experimental reproduction of clinical PMWS in gnotobiotic pigs and conventional pigs with tissue homogenates from pigs with naturally occurring PMWS and with cell culture propagated PCV2 produced mixed results. Clinical PMWS was reproduced in gnotobiotic (SPF) pigs and colostrum-deprived and caesarian-derived pigs co-infected with PCV2 and porcine parvovirus (PPV) (G. M. Allan et al., “Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11 (1999); S. Krakowka et al., “Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus,” Vet. Pathol. 37:254-263 (2000)), and in PCV2-inoculated gnotobiotic pigs when their immune system was activated by keyhole hemocyanin in incomplete Freund's adjuvant (S. Krakowka et al., “Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2),” Vet. Pathol. 38:31-42 (2001)).
Clinical PMWS was also reproduced in cesarean derived/colostrum deprived pigs (CD/CD) inoculated with PCV2 alone (P. A. Harms et al., “Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus,” Vet. Pathol. 38:528-539 (2001)) and in conventional pigs co-infected with PCV2 and either porcine parvovirus (PPV) or porcine reproductive and respiratory syndrome virus (PRRSV) (A. Rivora et al., “Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2,” J. Virol. 76: 3232-3239 (2002)). In cases of the PRRSV/PCV2 co-infection, the PMWS characteristic pathological signs such as lymphoid depletion, granulomatous inflammation and necrotizing hepatitis are induced by PCV2 and not by PRRSV (P. A. Harms et al., 2001, supra). However, clinical PMWS was not reproduced in gnotobiotic pigs infected with PCV2 alone (G. M. Allan et al., “Experimental infection of colostrums deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch. Virol. 145:2421-2429 (2000); G. M. Allan et al., “A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation, J. Vet. Med. 47:81-94 (2000); G. M. Allan et al., “Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11 (1999); M. Balasch et al., 1999, supra; J. Ellis et al., “Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets,” J. Vet. Diagn. Invest. 11:3-14 (1999); S. Kennedy et al., “Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus” J. Comp. Pathol. 122:9-24 (2000); S. Krakowka et al., 2001, supra; S. Krakowka et al., 2000, supra; R. M. Pogranichnyy et al., “Characterization of immune response of young pigs to porcine circovirus type 2 infection,” Viral. Immunol. 13:143-153 (2000)). The virus inocula used in these studies were either homogenates of tissues from pigs with naturally occurring PMWS, or virus propagated in PK-15 cell cultures (G. M. Allan et al., “Experimental infection of colostrums deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch. Virol. 145:2421-2429 (2000); G. M. Allan et al., “A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation, J. Vet. Med. 47:81-94 (2000); G. M. Allan et al., “Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11 (1999); M. Balasch et al., 1999, supra; J. Ellis et al., 1999, supra; S. Kennedy et al., 2000, supra; S. Krakowka et al., 2001, supra; S. Krakowka et al., 2000, supra; R. M. Pogranichnyy et al., 2000, supra). Since tissue homogenates may contain other common swine agents such as PPV and porcine reproductive and respiratory syndrome virus (PRRSV) (G. M. Allan et al., “Experimental infection of colostrums deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch. Virol. 145:2421-2429 (2000); G. M. Allan et al., “Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11 (1999); G. M. Allan and J. A. Ellis, 2000, supra; J. A. Ellis et al., “Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome,” J. Vet. Diagn. Invest. 12:21-27 (2000); C. Rosell et al., 2000, supra), and since the ATCC PK-15 cell line used for PCV2 propagation was persistently infected with PCV1 (G. C. Dulac and A. Afshar, 1989, supra), the clinical disease and pathological lesions reproduced in those studies may not be solely attributable to PCV2 infection (G. M. Allan et al., “Experimental infection of colostrums deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication,” Arch. Virol. 145:2421-2429 (2000); G. M. Allan et al., “A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation, J. Vet. Med. 47:81-94 (2000); G. M. Allan et al., “Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus,” J. Comp. Pathol. 121:1-11 (1999); G. M. Allan and J. A. Ellis, 2000, supra; J. A. Ellis et al., 2000, supra).
Clinical PMWS has also been reproduced in PCV2-inoculated CDCD pigs when vaccinated with Mycoplasma hyopneumoniae (G. M. Allan et al., “Immunostimulation, PCV-2 and PMWS,” Vet. Rec. 147:171-172 (2000)). Two recent field studies by G. M. Allan et al., “Neonatal vaccination for Mycoplasma hyopneumoniae and postweaning multisystemic wasting syndrome: a field trial,” Pig J. 48:34-41 (2001), and S. C. Kyriakis et al., “The effects of immuno-modulation on the clinical and pathological expression of postweaning multisystemic wasting syndrome,” J. Comp. Pathol. 126:38-46 (2002), tested the effect of immuno-modulation by Mycoplasma hyopneumoniae vaccine on the development of PMWS in endemic herds, and showed a significant decrease in PMWS cases in unvaccinated groups compared to the vaccinated animals. However, another recent study using conventional SPF piglets under controlled laboratory conditions could not reproduce such an effect, suggesting that vaccinations with M. hyopneumoniae may potentially influence the development of clinical PMWS but it is clearly a secondary role to a PCV2 infection. Based on these and other studies, PCV2 is nevertheless considered to be the primary but not the exclusive causative agent of PMWS.
The lack of an infectious virus stock of a biologically pure form of PCV2 has impeded the understanding of PCV2 pathogenesis and the etiological role of PCV2 in PMWS. Vaccinations against PPV and possibly PRRSV have not consistently been shown to prevent the onset of PMWS in PCV2 infected pigs. Consequently, finding a safe yet potent vaccine that specifically targets PMWS has been difficult. There is a definite art-recognized need in the veterinary field to produce an efficacious, safe vaccine against PCV2 infections and PMWS.
U.S. Pat. No. 6,287,856 (Poet et al.) and WO 99/45956 concern nucleic acids from psittacine beak and feather disease virus (BFDV), a circovirus that infects avian species, and from porcine circovirus (PCV). The patent proposes vaccine compositions comprising naked DNA or mRNA and discloses a nucleic acid vector for the transient expression of PCV in a eukaryotic cell comprising a cis-acting transcription or translation regulatory sequence derived from the human cytomegalovirus immediate or early gene enhancer or promoter functionally linked to a nucleic acid of the sequence. However, since the PCV DNA is derived solely from the PK-15 cell line, it is likely to comprise the nonpathogenic PCV1 discovered nearly 30 years ago by I. Tischer et al., 1974, supra, and, therefore, it is not likely to be effective in eliciting an immune reaction to PCV2 or infections caused by PCV2. Subunit vaccines of recombinant proteins made from vectors comprising open reading frames are also suggested in the patent but the open reading frames from PCV are not well characterized or distinguished from each other. Since the source of the PCV DNA is PK-15 cells, the proteins made from those vectors comprising the open reading frames of PCV1 would not possess reliable immunogenic properties, if any, against PCV2.
U.S. Pat. No. 6,217,883 (Allan et al.) and French Patent No. 2,781,159B relate to the isolation of five PCV strains from pulmonary or ganglionic samples taken from pigs infected with PMWS in Canada, California and France (Brittany), and their use in combination with at least one porcine parvovirus antigen in immunogenic compositions. Proteins encoded by PCV2 open reading frames (ORF) consisting of ORF1 to ORF13 are broadly described in the patent but there is no exemplification of any specific protein exhibiting immunogenic properties. The patent further discloses vectors consisting of DNA plasmids, linear DNA molecules and recombinant viruses that contain and express in vivo a nucleic acid molecule encoding the PCV antigen. Several other references, for example, U.S. Pat. No. 6,391,314 B1; U.S. Pat. No. 6,368,601 B1; French Patent No. 2,769,321; French Patent No. 2,769,322; WO 01/96377 A2; WO 00/01409; WO 99/18214; WO 00/77216 A2; WO 01/16330 A2; WO 99/29871; etc., also describe the administration of PCV1 or PCV2 polypeptides or the nucleic acids encoding the polypeptides of various strains.
However, the nonpathogenic PCV1 will not be useful against PCV2 infections and the pathogenic PCV2 strains described in the art, even if attenuated, are likely to be of limited value due to the usual tendency of a live virus to revert to its virulent state. Therefore, there is still a long-standing need in the art for a live, infectious, nonpathogenic antigen for the inoculation of pigs against serious infection or PMWS caused by PCV2 that is efficacious and remains safe in veterinary vaccines. These goals are met by the construction of the new live chimeric porcine circovirus described herein, which is based upon the genomic backbone of the nonpathogenic PCV1 isolated by I. Tischer et al. almost 30 years ago. The novel chimeric porcine circovirus of the present invention is able to satisfy that long-standing need by uniquely and advantageously retaining the nonpathogenic phenotype of PCV1 but eliciting specific immune response against pathogenic PCV2.